International Journal of Systematic Bacteriology (1 998),48,127-1 39 Printed in Great Britain
Comparison of PCR-based DNA fingerprinting
techniques for the identification of Listeria
species and their use for atypical Listeria
Mario Vaneechoutte,’ Patrick Boerlin,’t Hans-VoIker Tichyt3
Elizabeth Bannerman,’ Birgit Jager3 and Jacques Bille’
Author for correspondence: Mario Vaneechoutte. Tel: +32 9 240 36 92. Fax: + 32 9 240 36 59.
e-mail : Mario .Vaneechoutte @rug. ac.be
1 Department of Four PCR-based DNA fingerprinting techniques were compared for their ability
Microbiology and to identify a t the species level a heterogeneous collection of isolates
Immunology, Blok A,
University Hospital, 9000 belonging to the six valid Listeria species. 16s rDNA-RFLP analysis identified all
Gent, Belgium species and 16s rDNA-SSCP analysis identified almost all species. Also, isolates
* Swiss National Center for with unusual biochemical characteristics and/or unusual antigenic composition
Listeriosis and WHO could be identified correctly. rRNA-intracistronic length polymorphism analysis
Collaborative Center for suffered from high intraspecific variability, a limited number of fragments per
Foodborne Listeriosis, 1011
Lausanne, Switzerland profile, and small length differences between the spacers of different species.
tRNA-intergenic length polymorphism analysis resulted in identification of all
3 TUV Energie und
Systemtechnik GmbH, isolates but one, when fluorescent DNA capillary electrophoresis was used
Abteilung Biologische such that fragment length differences of 1 bp could be resolved. The four
Sicherheit, Freiburg i. Br., techniques yielded comparable results relevant to the taxonomy of Listeria.
They all indicate a high degree of genetic relatedness between L. innocua and
L. welshimeri, homogeneity of L. grayi, distinct but clear relatedness of L. grayi
to the other Listeria species, a clear distinction between the two subspecies of
L. ivanovii, and a clear distinction between Listeria isolates and isolates from
closely related taxa or from species which are phenotypically difficult to
distinguish from Listeria. New sequence determination of t h e 16s rRNA gene
was necessary to obtain sequences in accordance with the findings of 165
Keywords: Listeria, PCR-based DNA fingerprinting, 16s rDNA-RFLP, 16s rDNA-
SSCP, spacer length polymorphism
INTRODUCTION ivanovii, L. innocua, L . welshimeri, L. seeligeri and L.
grayi). The former species ‘ L . murrayi’ has been
The genus Listeria is very homogeneous, except for L . assigned to the species L. grayi (33). Only L. mono-
grayi which is clearly distinct from the other species (4, cytogenes and L. ivanovii are recognized as significant
7,22, 30, 34). The taxonomy of the genus Listeria has pathogens of humans and animals. The Listeria species
been difficult to assess in the past, but molecular differ in only a few phenotypic characteristics (12, 22,
methods (4, 30, 32) have clearly demonstrated the 31) and this high degree of similarity is the source of a
existence of six Listeria species (L. monocytogenes, L. lot of confusion in their identification. Furthermore,
several other species, such as Bacillus species, Entero-
......................I.............. 1 ................................................................................... .
I. ..................... coccus species (particularly Enterococcus faecalis)
t Present address: Department of Pathobiology, University of Guelph,
and Cellulomonas (Oerskovia) turbata, are often
Guelph, Ontario, Canada NIG 2W1. misidentified as Listeria species by inexperienced
Abbreviations: ARDRA, amplified rDNA restriction analysis; SSCP, single microbiologists. Finally, the type strain of L. mono-
strand conformational polymorphism. cytogenes is phenotypically abnormal (23) and would
The EMBL accession numbers for the sequences reported in this paper are be difficult to identify if it was encountered as a field
00576 0 1998 IUMS 127
M. Vaneechoutte and others
Methods which combine the reliability of genotypic supernatant of this boiled cell suspension was used as the
identification with the speed and simplicity of pheno- target DNA and was added - using filter-protected tips - to
typic methods such as biochemistry and serology, 45 pl aliquots of PCR mix for all DNA fingerprinting
would be more than welcome, particularly in the techniques.
presence of biochemically and serologically atypical Amplification mixture. All reaction mixtures contained - at
isolates. Four PCR-based DNA fingerprinting a final reaction volume of 50 pl - 0.005 U Goldstar poly-
methods have been described to enable identification merase pl-l (Eurogentec), 100 pM each dNTP (Pharmacia
of cultured bacterial organisms to the species level on Biotech) and 0.2-0.5 pM each primer in reaction buffer
the basis of genotype: rDNA-RFLP analysis (or [7.5 mM Tris/HCl, 2.5 mM MgCl,, 20 mM (NH,),SO,, 0.1 %
A R D R A , amplified rDNA restriction analysis) and (w/v) Tween 20, pH 9-01.After aliquotting in 45 pl volumes,
mixtures were overlaid with 40 pl mineral oil.
rDNA-single-strand conformational polymorphism
(SSCP) analysis, which both make use of the species- ARDRA. The 16s rRNA gene (16s rDNA) was amplified
specific sequence information in the 16s rRNA gene; using 0.2 pM primers complementary to conserved regions
and rDNA-spacer-PCR and tDNA-spacer-PCR, present at the edges of the 16s rDNA. The sequences of the
which have been reported to provide species-specific primers were S'TGGCTCAGATTGAACGCTGGCGGC
(Escherichia coli nucleotide positions 10-27) and 5'TAC-
length polymorphism of rRNA intracistronic and CTTGTTACGACTTCACCCCA (E. coli nucleotide pos-
tRNA-intergenic spacer regions, respectively. In this itions 1507-1485). After initial denaturation at 95 "C for
study we assessed the suitability of these four methods 5 min, the reaction mixtures were cycled 35 times for 45 s at
for the identification of the species of the genus 95 "C, 45 s at 55 "C, and 1 rnin at 72 "C. Finally, a 7 rnin
Listeria. extension period at 72 "C was carried out. The presence and
yield of specific PCR products were determined by Multi-
ARDRA has been studied most thoroughly and has Purpose (Boehringer) agarose (1 %, w/v) - ethidium bro-
been used for the identification of species of different mide (50 ng ml-l) gel electrophoresis for 30 rnin at 7 V cm-l.
taxa, e.g. Acinetobacter (39), Clostridium (37), Coma- Ten microlitre aliquots of the amplified DNA were used
monadaceae (4 l), Corynebacterium (40), Mollicutes without further purification for restriction digestion. The
(9), Mycobacterium (38), Leptospira (29) and Strep- following enzymes were used: AluI, AvaI, BanI, BfaI,
tococcus (19), and to study phylogenetic relationships Bsp12861, BstEII, BstUI, CfoI (HhaI isoschizomer), DraI,
in Bacillus (16), Clostridium (13), Moraxella (18) and EcoT381, HaeIII, HinfI, MboI, MspI, MvaI, NciI, RsaI,
Xanthomonas (28). Sau961, ScrFI, StyI, TaqI and Tru9I. Restriction digestion
was carried out for 1 h at 37 "C, except for TaqI and Tru91,
Widjojoatmodjo et al. (44, 45) used SSCP analysis of which were incubated at 65 "C, in 20 pl volumes of com-
part of the 1 s rDNA to identify a number of species, mercial incubation buffer containing 1 U p1-l restriction
including all six Listeria species. Jensen et al. (20) enzyme and 5 pl amplified 16s rRNA gene product. Re-
showed that rDNA-spacer-PCR could be used to striction fragments were separated by gel electrophoresis for
identify Escherichia species, some Enterobacter and 3 h at 7 V cm-l on 3 % Metaphor agarose (FMC Bio-
Staphylococcus species and five of the six Listeria Products Europe) containing 50 ng ethidium bromide ml-'.
species (L. seeligeri was not studied). Others used this 165 rDNA-SSCP analysis. rDNA-SSCP analysis was carried
approach successfully for the identification of Legion- out essentially as described by Widjojoatmodjo et al. (44).
ellaceae (17). Primers P l l P (5'GAG GAA GGT GGG GAT GAC GT)
and P13P (5'AGG CCC GGG AAC GTA TTC AC) were
tDNA-spacer-PCR has been used for the identification used at concentrations of 0.2 pM to amplify a 216 bp
of Staphylococcus species (42, 43), Streptococcus fragment of the V6 region of the 16s rDNA ( E . coli 16s
species (26) and Acinetobacter species (10, 46). rDNA positions 1 175-1 390). After initial denaturation at
95 "C for 5 min, the reaction mixtures were cycled 35 times
METHODS for 20 s at 95 "C, 1 rnin at 55 "C and 1 rnin at 72 "C. Finally,
a 7 rnin extension period at 72 "C was carried out: 0-3 pl
Bacteria. Table 1 lists the isolates which were used. Besides volumes of the amplification products were mixed with 6 pl
representatives of all the six valid Listeria species, including water and 3 pl sequencing buffer (95 YO formamide, 20 mM
the two subspecies of L. ivanovii (3), isolates of the species L. EDTA, 0.05% bromophenol blue and 0.05% xylene
monocytogenes, L. innocua, L. welshimeri and L. ivanovii cyanol). The mixture was heated to 95 "C for 5 min and
representing unusual biotypes or atypical antigenic com- loaded on the gels. The amplification products were separ-
positions were also included and tested blindly. The Listeria ated in non-denaturing 0.5 x MDE gels (FMC BioProducts)
isolates representing atypical biochemical or serological containing 5 % glycerol. The gels were run overnight in a
reactions were also tested with API-Listeria galleries Protean I1 xi electrophoresis cell (Bio-Rad) in 0.5 x TBE at
(BioMerieux), following the recommendations of the manu- room temperature at 90 V. The gels were stained using the
facturer. Isolates of genera phylogenetically closely related Silver Stain Plus kit (Bio-Rad) and the profiles were
to Listeria were also included since these may be pheno- interpreted visually.
typically misidentified as Listeria. rRNA-intracistronic spacer length polymorphism analysis
Sample preparation for PCR. Target DNA was prepared from (rDNA-spacer-PCR). The intracistronic rDNA spacer regions
cells grown overnight at 37 "C on Mueller-Hinton Agar I1 were amplified by using primers - at concentrations of
(MHAII ; BBL Microbiology Systems) supplemented with 0.5 pM - flanking the 16s-23s rDNA spacer region (for-
5 % sheep blood. A 1 p1 volume of cells was suspended in ward, 5'GAA GTC GTA ACA AGG; reverse, 5'CAA GGC
300 pl 10 mM Tricine/l.5 mM MgC1, and heated at 100 "C ATC CAC CGT) (20). After initial denaturation at 95 "C for
for 10 rnin in a heating block. Five microlitres of the 5 min, the reaction mixtures were cycled 25 times for 1 min
128 International Journal of Systematic Bacteriology 48
PCR fingerprinting for identification of Listeria species
at 94 "C, 2 rnin at 55 "C, and 2 rnin at 72 "C, followed by a
final 7 rnin extension period at 72 "C. Twenty microlitres of
each amplification product was separated in 4 % poly-
acrylamide gels using 1 x TBE at constant voltage in a
Protean I1 xi electrophoresis cell. The gels were stained in a
1 pg ethidium bromide ml-I solution and photographed
under short wavelength UV light. The migration profiles
were compared visually.
tRNA-intergenicspacer length polymorphismanalysis (tDNA-
spacer-PCR). tDNA-spacer-PCR was carried out essentially
as described by Welsh & McClelland (42). Primers T5A
(5'AGT CCG GTG CTC TAA CCA ACT GAG) and T3B
(5'AGG CCG CGG GTT CGA ATC C) were used at
concentrations of 0.5 pM to amplify the spacer regions
between the tRNA genes. For analysis by capillary electro-
phoresis the T3B primer was labelled at the 5'end with the
fluorescent amidite TET (Applied Biosystems). After initial
heating at 95 "C for 7 min, reaction mixtures were cycled 40
times for 30 s at 95 "C, 30 s at 50 "C and 2 rnin at 72 "C,
followed by final extension for 7 rnin at 72 "C. Capillary
electrophoresis of fluorescently labelled PCR products was
done on an ABI 310 (Applied Biosystems) using the POP-4
polymer and capillaries and with the TAMRA-500 standard
as the internal lane size standard.
165 rDNA sequence determination. An approximately
1.45 kb segment of the 16s rRNA gene was amplified using
primer 41f (5'GCT CAG ATT GAA CGC TGG CG) and
the biotin-labelled primer 14881-(S'biotin-CGG TTA CCT Fig, 1. 165 rDNA-RFLP profiles obtained by 3 % agarose gel
electrophoresis of digests of the amplified 165 rDNA after
TGT TAC GAC TTC ACC). Forty microlitres of the PCR restriction with Alul .
sample were added to Dynabeads M280 Streptavidin
(Dynal), the DNA was denatured and the biotin-labelled
single strand was immobilized onto the beads and used as
one template for the sequencing reaction using fluorescent BstUI, CfoI, RsaI and Tru9I enabled some differen-
primers 358f (5' AGA CTC CTA CGG GAG GCA GCA tiation (Table 1). Combination of the profiles observed
GT), 536f [5' GTG CCA GC(AC) GCC GCG GTA ATA C] after digestion with AluI (Fig. 1) and BstUI enabled
and 928f [5' TAA AAC T(CT)A AA(GT) GAA TTG ACG identification of all Listeria species, except for L.
GGG]. The opposite strand remaining in solution was also innocua and L. welshimeri, for which a constant
used as template using fluorescent primers 336r [5' ACT difference could be shown only after restriction of the
GCT GCS (CT)CC CGT AGG AGT CT] and 51.51-[5' 16s rDNA with Tru9I (Table 1, Table 2). Also, only
G(AT)A TTA CCG CGG C(GT)GCTG GCA C]. minor but constant differences could be observed for
Sequencing reaction products obtained by using the T7 BstUI restriction profiles of L. ivanovii subsp. ivanovii
Autoread kit (Pharmacia Biotech) were processed using an
ALF DNA sequencer (Pharmacia Biotech) and the ALF and L . ivanovii subsp. londoniensis, and for L.
manager software. DNA sequence comparisons were done welshimeri and L . seeligeri (Table 2). L. grayi was
using the HIBIO DNASIS software package (Hitachi). clearly different from the other five species, since
Published Listeria sequences were obtained from the EMBL restriction with AluI and RsaI yielded unique re-
database and the database included in the ARB package (see striction profiles not observed for any other Listeria
below). Tree construction was done using the ARB software species (Table 1, Table 2). One L. innocua isolate
package developed by Oliver Strunk, Wolfgang Ludwig and (TI6520) had a BstUI profile (lc) with an additional
others from the Microbiology Department of the Technical 250 bp fragment. For L. monocytogenes isolate I P l l
University, Munich, Germany. The software version for an extra fragment was observed repeatedly in the CfoI
Linux and the database (6aug9.ascii.arb) were obtained by profile. This could be resolved as the result of in-
anonymous ftp from ftp.biol.chemie.tu-muenchen.de.
terference of minor non-specific amplification, not
Computer-assisted restriction analysis of the 16s rRNA present in one of the collaborating laboratories. The
gene was carried out with HIBIO DNASIS on EMBL isolates of the closely related non-Listeria species could
sequences X56148-X56154 (7) and on the sequences ob- easily be identified as such by restriction analysis of the
tained in the present work. Distance values were calculated
using the neighbour-joining method with Kimura correc- 16s rDNA, although some of the restriction enzymes
tion. yielded identical profiles for some Listeria and non-
Listeria species (Table 1).
16s rRNA gene sequence determination and
ARDRA phylogenetic tree construction for the genus Listeria
Restriction of the 1 s rRNA gene was carried o u t with T h e sequences of the 16s rRNA genes were determined
a total of 22 enzymes. Only restriction with AluI, for seven isolates (indicated in Table 1). The distance
International journal of Systematic Bacteriology 48 129
M. Vaneechoutte and others
Table 1. Designation of isolates, serotype and biotype, and of profiles observed for different PCR-based DNA
fingerprinting techniques for species identification
Original no. Species* Serotype or unusual
RsaI BstUI Tru9I
1 LL1955 L. monocytogenes 4b 1 1 1 la la 1 6
2 ZH7415 L. monocytogenes 1/2a 1 1 1 la la 1 4
3 SLCC2371 L. monocytogenes 1/2a 1 1 1 la ND 1 4
4 SLCC2755 L. monocytogenes 1/2b 1 1 1 la ND 1 5
5 SLCC2372 L. monocytogenes 1/2c 1 1 1 la ND 1 4
6 SLCC2373 L. monocytogenes 3a 1 1 1 la ND 1 4
7 SLCC2540 L. monocytogenes 3b 1 1 1 la ND 1 4
8 SLCC2479 L. monocytogenes 3c 1 1 1 la ND 1 4
9 SLCC2374 L. monocytogenes 4a 1 1 1 la la 1 9
10 SLCC2375 L. monocytogenes 4b 1 1 1 la ND 1 7
11 SLCC2376 L. monoeytogenes 4c 1 1 1 la ND 1 10
12 SLCC2377 L. monocytogenes 4d 1 1 1 la ND 1 4
13 SLCC2378 L. monocytogenes 4e 1 1 1 la ND 1 6
14 SLCC2482 L. monocytogenes 7 1 1 1 la ND 1 5
58 IP11 L. monocytogenes V/Vl ;VI; V1I;VIII ;A ;B; C 1 1 ND la la 1 8
59 IP15 L. monocytogenes" 4b 1 1 ND la ND 1 ND
65 Nv3953 L. monocytogenesb 1/2b 1 1 ND la ND 1 ND
66 NV4 172 L. monocytogenesb 1/2a 1 1 ND la ND 1 ND
68 LU4526 L. monocytogenesC 4b 1 1 ND la ND 1 ND
77 BS6179 L. monocytogenes" 4b 1 1 ND la ND 1 ND
79 GE6366 L. monocytogenes' 1/2c 1 1 ND la ND 1 ND
15 SLCC3379/ATCC 330905 L. innocua 6a 2 1 1 la la 2 13
16 LL271 L. innocua 6b 2 1 1 la la 2 11
17 VD8304 L. innocua 6a 2 1 1 la la 2 12
18 8716 L. innocua 6b 2 1 1 la la 2 11
25 VD7945 L. innocua 6b 2 1 1 la la 2 11
69 GE5432 L. innocua I; I/II;A; B;C (1/2b) 2 1 ND la la 2 ND
70 GE5433 L. innocua I; I/lI;A; B;C (1/2b) 2 1 1 la la ND ND
71 TI5796 L. innocua V/VI ;VII ;XV; B; C ; D 2 1 ND la la 2 ND
76 VD6 140 L. innocua V/VI;VII;XV;B;C;D 2 1 1 la la ND ND
81 TI6520 L. innocua XV ;B ;C ;D 2 1 ND lc la 2 ND
82 TI6551 L. innocua V/VI ;VII ;XV: B ;C ; D 2 1 1 la la ND ND
83 BL6621 L. innocua No somatic factors A;B;C 2 1 1 la la ND ND
87 BL6830 L. innocua VII; VI1I;A; B; C 2 1 ND la la 2 ND
23 SLCC5334 L. welshimeri 6a 2 1 1 la lb 2 14
24 ZH7199 L. welshimeri 6b 2 1 1 la lb 2+3 14
60 LL244 L. welshimeri V/VI ;VI ;VII ;X ;B ;C ;D 2 1 ND la lb 2+3 ND
61 LL519 L. welshimeri I;I/II; A; B; C (1/2b) 2 1 ND la lb 2 ND
62 LL520 L. welshimeri I;I/II;A;B;C (1/2b) 2 1 ND la lb 2 ND
63 BS2094 L. welshimeri V/VI;VI;VII;X;B;C;D 2 1 ND la lb 2+3 ND
67 SO4352 L. welshimeri V/VI;VI: VI1;X: B;C; D 2 1 1 la lb ND ND
72 TI5800 L. welshimeri V/VI;VI;VII;X;B;C;D 2 1 ND la lb 2+3 ND
73 GE5877 L. weishimeri V/VI; VI: V1I;X: B;C; D 2 1 1 la lb ND ND
74 BE5963 L. welshimeri V/VI ;VI: V1I;X: B; C ; D 2 1 1 la lb ND ND
75 BE6108 L. weishimeri V/Vl;VI;VII;X; no H-factors 2 1 1 la lb ND ND
78 GE6254 L. welshimeri V/VI ;VI: V1I;X: B; C ; D 2 1 1 la lb ND ND
84 LU6662 L. welshimeri V/VI ;VI; VII ;X; B ;C; D 2 1 ND la lb 2+3 ND
85 BE61 12 L. welshimeri V/VI ;VI: VI1;X: B; C; D 2 1 1 la lb ND ND
86 AG7166 L. welshirneri V/VI; VI: VI1;X: B; C; D 2 1 1 la ib ND ND
19 SLCC3954/ATCCC 359678 L. seeligeri 1/2b 2 2 1 lb la 3 3
20 8375 L. seeligeri 4ab 2 2 1 lb la 3 1
21 BE8950 L. seeligeri 1/2b 2 2 1 lb ND 3 1
22 BE8953 L. seeligeri 4d 2 2 1 lb ND 3 2
64 NV2918 L. seeligerid 1/2b 2 2 m l b ND 3 ND
27 LL278 L . ivanovii subsp. ivanovii 5 2 2 1 2a la 6 ND
28 LL483 L. ivanovii subsp. ivanovii 5 2 2 1 2a la 6 21
29 BE5087 L . ivanovii subsp. ivanovii 5 2 2 1 2a ND 6 21
46 CLIP 125lOT§ L. ivanovii subsp. ivanovii 5 2 2 1 2a ND 6 21
51 SLCC4306 L . ivanovii subsp. ivanovii 5 2 2 1 2a ND 6 20
55 SLCC2098 L. ivanovii subsp. ivanovii 5 2 2 1 2a ND 6 21
30 BE1604 L. ivanovii subsp. londoniensis 5 2 2 1 2b la 4 19
31 BE5063 L. ivanovii subsp. londoniensis 5 2 2 1 2b la ND ND
32 BE1694 L. ivanovii subsp. londoniensis 5 2 2 1 2b ND 4 ND
33 BE3728 L. ivanovii subsp. londoniensis 5 2 2 1 2b ND 4 ND
47 CLIP12229T5 L. ivanovii subsp. londoniensis 5 2 2 1 2b la 4 18
48 BE5194 L. ivanovii subsp. londoniensis I;I/II;A;B;C (1/2b) 2 2 1 2b ND 4 ND
50 BE5195 L. ivanovii subsp. londoniensis I/II;VII;no H-factors 2 2 1 2b ND ND ND
130 International Journal of Systematic Bacteriology 48
PCR fingerprinting for identification of Listeria species
Table 1 (cont.)
L. ivanovii subsp. londoniensis
L. ivanovii subsp. londoniensis
Serotype or unusual
I; I/II;A; B ; C (1/2b)
43 ATCC 2540211 L. grayi 3 2 2 2a 2 5 17
44 ATCC 2540311 L. gra-vi 3 2 2 2a 2 5 15
45 SLCC2080/ATCC 191208 L . grayi 3 2 2 2a ND 5 16
34 LA3056 Brochothrix thermosphacta 4 3 2 1 ND ND ND
35 LA3674 Bacillus cereus 5 2 3 2b ND ND ND
39 CIP 103610T Carnobacterium gallinarum 9 6 4 4 N D ND ND
42 LA3193 Cellulomonas (Oerskovia) turbata 11 8 6 ~ N D
36 LA2365 Enterrococcus durans 6 4 4 4 N D ND ND
37 LA2390 Enterococcus jaecalis 7 4 4 4 N D ND ND
38 LA3225 Jonesia (Listeria) denitri$cans 8 5 5 5 ND ND ND
40 CIP 102976T Vagococcusfluvialis 10 7 4 3 ND ND ND
ND, Not determined.
* Superscript letters indicate the following properties : a, arabitol-negative ; b, L-rhamnose-negative ; c, methyl a-D-mannoside-
negative ; d, methyl a-D-mannoside-positive.
t Serovar numbering and serological reactions are according to Seeliger & Hohne (35). Serotypes with flagellar factors B; C ; D or
somatic factors VII ;VIII or I ;1/11;VII have not been described previously.
$ rDNA-SSCP analysis was at room temperature.
§Isolates of which the 16s rRNA gene was sequenced in this study; their EMBL accession numbers are X98526X98532.
11 These isolates were formerly ‘L. murruyi’.
values are given in Table 3 and a tree representation of londoniensis and L. grayi) had specific and homo-
this calculation is shown in Fig. 2. The sequences geneous profiles. The eight L. welshimeri isolates
obtained in the present study differ from those ob- presented two different profiles, and for three of these
tained previously (7,8). The number of differences (not isolates this profile was identical to the unique L.
counting undefined bases as differences and comparing innocua profile (Table 1). Electrophoresis was also
with the best corresponding to previously published performed under different conditions (four different
sequences for each species) was between 3 bp (with L. buffer systems, electrophoresis at 6 “C or at room
seeligeri sequence X56148) and 8 bp (with L. grayi temperature, addition of 0 or 10% glycerol). The
sequence X56150). To construct a tree based on the profiles obtained varied markedly, depending on the
16s rRNA sequences of the Listeria species studied conditions used, but some or all L. welshimeri isolates
here, they were imported into the ARB package and remained indistinguishable from L. innocua, whereas
aligned manually to match the alignment of the differentiation of other species became difficult or
Listeria sequences determined previously (7, 8). A impossible (data not shown).
phylogenetic tree containing the published sequences
and a combined tree containing also the sequences rDNA-spacer-PCR
obtained in this study were constructed (data not Analysis of the PCR products by PAGE showed that
shown). The clustering of the species in all three trees 21 profiles could be found among the 37 isolates tested
did not show significant differences.Thus, the sequence (Fig. 4, Table 1). Several profiles were found within
discrepancies found between published and new se- each species, except for L. welshimeri (for which only
quences had no influence on the determination of two isolates were tested). The profiles were composed
phylogenetic relationships, although there were less of one main band and one or several minor bands. The
interspecific differences for the newly determined variation within a species relied on both the minor and
sequences. Computer-aided restriction digestion of the the main bands. Because of the variability within the
newly determined sequences corresponded perfectly species it was difficult to define reliable diagnostic
with the observed ARDRA patterns, except for two criteria for species identification. rDNA-spacer-PCR
minor discrepancies (Table 2). was clearly reproducible. When repeating electro-
phoresis with new amplification products from the
rDNA-SSCP analysis same strains, the same pofiles were obtained and the
same differences between profiles were reproducibly
The results of the rDNA-SSCP analysis are reported in detectable.
Table 1 and are illustrated in Fig. 3. Seven profiles were
obtained for the six Listeria species examined. The t DNA-spacer-PCR
profiles were composed of two or four bands. Five
Listeria species and subspecies ( L . monocytogenes, L. The discriminatory power of tDNA-spacer-PCR de-
seeligeri, L. ivanovii subsp. ivanovii, L. ivanovii subsp. pended highly on the resolving power of the electro-
International Journal of Systematic Bacteriology 48 131
Table 2. Comparison of observed ARDRA patterns with restriction fragment sites and lengths as predicted by 165 rRNA sequence determination
Restriction with Restriction sites Restriction fragment lengths ARDR
3213 127 140 207 822/3 98213 1029/30 1234/5 13 3213 47 67 95 160 175 205 23213 438 61516
L. monocytogenes + + + + + + + + + + + + +
L. innocua + + + + + + + + + + +
L. welshimeri + + + + + + + + + + +
L. seeligeri + + + + + + + + + + +
L. ivanovii ivanovii + + + + + + + + + + +
L. ivanovii londoniensis + + + + + + + + + + +
L. gmyi + + + + + + + + + + + + + + + + +
CfOI (CCCIC) 145 53718l9140 723/4 1069170 2 145 182 346 392 395 53718
L. monocytogenes + ++ + + - H + + + + + 1
L. innocua + ++ + + + + + + + + + 1
L. welshimeri + - H + + + + + + + + + 1
L. seeligeri - H + + ++ + + + + 2
L. ivanovii ivanovii ++ + + ++ + + + + 2
L. ivanovii Iondoniensis ++ + + ++ + + + + 2
L. gmyi + + t + + ++ + + + + 2
BsiUI (CGICG) 196 362 487 537 539 72314 93213 121718 1224/ 131112 13261 2 718 15 50 87 94 125 14011 166 18415 196/7 209 24213 285 29213
L. monocytogenes + + + + + + + + + + + + + + + + + + + la
L. innocua + + + + + + + + + + + + + + + + + + + la
L. welshimeri + + + + + + + + + + + + + + + + + la*
L. seeligeri + + + + + + + + + + + + + + + + + lb
L. ivanovii ivanovii + + + + + + + + + + + + + + + + + + + + + + + 2a
L. ivanovii londoniensis + + + + + + + + + + + + + + + + + + + + + 2b
L. gmyi + + + + + + + + + + + + + + + + + + + + + 2a
nu91 (TITAA) 553 579/80 831 837 917 919 1051/2 1076 25/6/7 8216 13314 252 257 391 415 553
L. monoqvtogenes + + + + + + + + + + + la
L. innocua + + + + + + + + + + + la
L. welshimeri + + + + + t + t + + + + + lb
L. seeligeri + + + + + + + + + + + la
L. ivanovii ivanovii + + + + + + + + + + + la
L. ivanovii londoniensis + + + + + + + + + + + lb
L. grayi + + + + + + + + + + + 2t
*According to the sequences determined in this study, L. welshimeri has a deletion which destroys the BstUI restriction site at position 1217. This predicts ARDRA pattern lb. However, for all L. welshimeri
strains ARDRA pattern 1a was observed.
?For L. gruyi, both the 252 and 257 bp fragments were observed on ARDRA patterns instead of the predicted 257 bp fragment only.
PCR fingerprinting for identification of Listeria species
Table 3. Distance values data matrix of the Listeria 165 rRNA gene sequences determined in this work
Values were calculated using the neighbour-joining method (Kimura correction, vertical gaps compressed) provided by the ARB
L. grayi L. innocua L. ivanovii subsp. L. monocytogenes L. seeligeri L. welshimeri
L. grayi 0.0000 0.0424 0.0348 0.0393 0-0386 0.0393 0.0401
L. innocua 0.0000 0.0 100 0.0093 0.0057 0-0071 0-0057
L. ivanovii subsp. ivanovii 0.0000 0.0035 0.0 122 0.0064 0.0 100
L. ivanovii subsp. londoniensis 0-0000 0.0 136 0.0043 0.0085
L. monocytogenes 0~0000 0.0114 0.0 100
L. seeligeri 0~0000 0.0057
L. welshimeri o*oooo
L. ivanovii .....................................................................................................
subsp. londoniensis subsp. ivanovii Fig. 2. Tree representation of distances
between the 165 rDNA sequences of Listeria
L. seeligeri species. Only the sequences as determined in
this study were used for the above
representation. E. coli K-12 was used as an
innocua L. welshimeri outgroup. The tree was constructed with the
built-in function of the ARB software
L. monocytogenes package using the neighbour-joining
method (Kimura correction, vertical gaps
cpm-pressed). The value for the distance
between E. coli and the Listeria cluster is
0.05 indicated by the arrows.
intraspecific differences were observed, several - inter-
PCR and inter-electrophoresis run - reproducible dif-
ferences between the species were present and enabled
clustering into groups corresponding with established
taxonomy (Table 4, Fig. 5). For instance, L. mono-
cytogenes isolates were characterized by the presence
of a 284, 285 or 286 bp peak, absent in all other
isolates. Two L. monocytogenes isolates [SLCC 2374
(LIS9) and SLCC 2376 (LIS1 l)] lacked this peak, but
could be differentiated from all other species by the
presence of a 265 and a 267 bp peak (Fig. 5). L. innocua
showed most intraspecific heterogeneity and had most
peaks in common with L. monocytogenes, from which
it could only be differentiated by the absence of the
284-286 and the 267 bp peaks. The profile of one L.
innocua isolate (TI6520) showed combined charac-
teristics of L. innocua, L. welshimeri and L. mono-
fig. 3. SSCP profiles of an amplified 216 bp fragment of the 165
rDNA as observed after an overnight run in non-denaturing
cytogenes profiles (Table 4), and thus, despite repeated
0-5 x MDE gel in 0.5 x TBE +5 % glycerol at room temperature testing, could not be identified. This is the same isolate
at 90 V and silver staining. with a slightly aberrant BstUI restriction pattern
(Table 1). All isolates of L. monocytogenes, L. innocua
and L. welshimeri had a tRNA spacer of 159 bp, while
this spacer was 160 bp long for all L. seeligeri isolates
phoresis technique used. Agarose gel electrophoresis and 162 bp (with sometimes an additional 161 bp
and 5 % PAGE were not sufficiently discriminatory for peak) for all L. ivanovii isolates. The subspecies of the
interpretation of the tDNA-spacer-PCR profiles (data latter species could be differentiated by a single bp
not presented). length difference of a tRNA spacer region of 85 bp for
L. ivanovii subsp. londoniensis and 86 bp for L. ivanovii
Only capillary electrophoresis provided sufficient res- subsp. ivanovii.-Isolates of species other than Listeria
olution to yield useful results. Although substantial had clearly different tDNA PCR profiles.
International Journal of Systematic Bacteriology 48 133
M. Vaneechoutte and others
Fig. 4. rDNA-spacer-PCR profiles as observed after 5 % PAGE and ethidium bromide staining (negative image).
For tDNA spacer amplification, reproducibility prob- This is particularly true for L. monocytugenes and L.
lems of the amplification step were observed. Upon innocua, which must be clearly distinguished because
analysis with agarose gel electrophoresis and PAGE, of the health hazard represented by the former but not
sometimes additional peaks were observed depending by the latter. However, both the haemolysis and the
on the PCR run and relative peak height was variable Christie-Atkins-Munch-Petersen (CAMP) test with
between PCR runs (data not shown). Similar problems Staphylococcus aureus, which are the distinctive cri-
have been observed with e.g. arbitrarily primed PCR teria for these two species, are not always easy to
(36). However, highly identical profiles for different interpret because some L. monocytogenes isolates
PCR runs were observed using capillary electro- produce only very faint haemolysis and/or weakly
phoresis. positive CAMP reaction. A positive CAMP reaction
with Rhodococcus equi has recently been proposed as
Aty picaI isolates an additional identification criterion for L. mono-
cytogenes (11). However, this test has long been
The molecular identification of atypical L. ivanovii controversial and has not been adopted by all lab-
subsp. londoniensis and of all the atypical L. mono- oratories. Its validity also heavily depends on the R.
cytogenes and L. seeligeri using ARDRA, rDNA- equi strain used for the reaction (1 1).
SSCP analysis (Table I), and tDNA-spacer-PCR
analysed on capillary electrophoresis was in full Several commercial galleries have recently been de-
agreement with the results obtained with the API veloped to make the identification of Listeria species
galleries (Table 1). The phenotypically atypical L. easier (1, 2, 24). Nevertheless, since some identifi-
innocua and L. welshimeri isolates could only be cations still rely on a unique characteristic (2) for
definitely identified by ARDRA and tDNA-spacer- which some isolates may show an ambiguous reaction,
PCR. additional tests like haemolysis and CAMP may still
be necessary for a correct identification (24). Fur-
thermore, biochemically abnormal isolates are regu-
larly encountered during routine identification of
Because of their high phenotypic similarity, only a Listeria isolates. Thus, in spite of tremendous improve-
limited number of biochemical tests can be used to ments in commercial kits, phenotypic identification
differentiate the Listeria species from one another (3 1). still remains difficult for a proportion of the isolates (1,
134 International Jo urna I of Systematic Bacteriology 48
PCR fingerprinting for identification of Listeria species
2). In such cases, rapid and reliable confirmation based
on genetic criteria remains of great interest.
We evaluated the effectiveness of four of the PCR
+ + > + I I I I I
fingerprinting techniques for the identification of
isolates of the genus Listeria. ARDRA enabled differ-
I I I I I I I IF entiation among all six species and between the two L .
ivanovii subspecies, although only minor differences
were detected between the non-haemolytic species L .
innocua and L. welshimeri and between both L . ivanovii
I I S 1 I I I I I subspecies.
High discriminatory power was also obtained with
rDNA-SSCP analysis (44), which identified all the
Listeria species, except L. innocua and some L.
I I I I I I + + + welshirneri isolates, which shared indistinguishable
profiles under several of the electrophoresis conditions
1 tested. Since the sequences of the 16s rRNA gene
+ I I I I I I I I
regions amplified for SSCP analysis diverge at only
one position for these two species, this lack of
I l l
discrimination is not unexpected. A more recent study
(45) showed that SSCP analysis of the rDNA required
I + I
I l l
two different primer sets to differentiate all six Listeria
species. Since six copies of the rRNA-operon are
I l l 0
1 I present in the genome of Listeria species (27), it is
possible that the four bands observed in some of the L.
+ l + l I I + I I welshimeri isolates rely on the simultaneous presence
of two distinct alleles of the rRNA operon in the
I I I + + + l I I genome of these particular isolates.
+ + + +
I I + + l
Jensen et a/. (20) reported that Listeria isolates can be
identified on the basis of the sizes of the 16s-23s
intracistronic rDNA spacers. The molecular sizes of
the major fragments obtained by this method in the
present work were approximately the same as those
originally described. However, we found on several
- occasions differences in the banding profiles of isolates
belonging to the same species, sometimes even in the
I I I I I I I I + major bands which have been suggested as diagnostic
* markers for species identification (Fig. 4). In our
+ + + + I + I I I hands, this technique was therefore not useful for easy
identification of the species of the genus Listeria,
because of the lack of species-specific bands within the
profiles of some species and because sometimes only
minor electrophoretic migration differences were ob-
+ + * I I + I I I I served between profiles of different species. The reason
I I I I I I I IF
for the discrepancy between both studies is not known,
I but the differences may be due to the use of different
populations of Listeria isolates. Although we ex-
amined less isolates than Jensen et al. (20), the isolates
used here were selected to cover a large spectrum of
serotypes and may represent a more heterogeneous
population than that used by these authors. Our results
suggest that the sequence and length variability of the
rDNA spacers may be greater than expected in the
genus Listeria and that, as in other species ( 5 , 14, 15,
25), rDNA-spacer-PCR may be used as a typing tool
rather than an identification tool. Jensen & Straus (21)
pointed to the need for using special amplification
conditions i.e. low concentration of polymerase and
polymerase buffer - to avoid multiple banding
patterns. However, we obtained identical profiles when
4 4 4 4 4 4 4 4 4 using these special conditions (data not shown).
International Journal of Systematic Bacteriology 48 135
M. Vaneechoutte and others
I I Ill 1
I I I II II LIS17inn
I 1 II II LIS16inn
1 1 II I1 LIS18inn
I I II I LIS25inn
I I II I LIS23wel
I I II I
I I II II I LI52 1see
I I 111 I LIS22see
I I II I LIS19see
1 I II I LIS28iviv
I I I1 I LIS29iviv
I I II II LIS27iviv
I I I1 I LIS3Oivlo
I I i II I LIS3livlo
I I I I Ill II
I I I I Ill I LIS13mon
I I I I Ill II LlS7mon
I I I I Ill I LIS14mon
I I I I Ill II LIS5mon
I I I I Ill I1 LIS6mon
I I I 1 Ill II LIS8mon
I I I I I I IIII II LlSl2mon
I I I I Ill Ill LIS3mon
I I I II Ill II LIS4mon
I I I Ill 111 LlSlmon
I I I Ill 111 LlSZmon
I I I IIII II LIS9mon
I I I Ill II LISllmon
Fig. 5. Clustering with Ward algorithm of tDNA-spacer-PCR profiles obtained by capillary electrophoresis on an ABI 310
apparatus. Calculation of similarity matrix based on band-matching using area-sensitive coefficient (tolerance band
position 0.1%, minimal surface bands: 0 % of total profile). Similarity indicated as a percentage on top of the
dendrogram; fragment length of amplified tRNA spacers indicated in bp on top of fingerprints. Abbreviations of strain
designations: inn, L. innocua; iviv, L. ivanovii subsp. ivanovii; ivlo, L. ivanovii subsp. londoniensis; mon, L.
monocytogenes; see, L. seeligeri; wel, L. welshimeri. Numbers in the strain designation abbreviations refer to the study
code as indicated in Table 1.
Capillary electrophoresis could reproducibly separate hand, L. ivanovii and L. grayi have species-specific
fragments differing by only a single base pair in length antigenic combinations and the biochemically very
and only this high resolution enabled sufficient dis- close species L. rnonocytogenes and L. innocua do not
crimination between the tDNA PCR profiles. En- share the same serovars and can therefore be differ-
hanced tDNA-PCR reproducibility by the use of entiated serologically. On the other hand, L. seeligeri
capillary electrophoresis may be explained by the fact cannot be serologically distinguished from L . mono-
that the high resolution offered by this electrophoresis cytogenes, nor can L. welshimeri be distinguished from
technique enables the detailed analysis of smaller sized L. innocua.
fragments (basically in the range 60-300 bp), which The species identification of 13 antigenically abnormal
may be more reproducibly amplified. Listeria isolates (Table I), as obtained with standard
However, the subtlety of the observed differences, the biochemical tests and with the API Listeria galleries,
peak height variability, the possible interference of was confirmed by ARDRA, rDNA-SSCP analysis and
background amplification products and the huge tDNA spacer analysis. Among others, these data show
intraspecific variability (as was also observed with 5 % that serovar 1/2b isolates can also be found in Listeria
PAGE) require careful evaluation of the practical species other than L. rnonocytogenes and L . seeligeri
applicability of tDNA-spacer-PCR. It can be con- (i.e. in L. innocua, L. ivanovii subsp. londoniensis and L.
cluded that thorough standardization of PCR con- welshirneri) and consequently, that serotyping may not
ditions, high-resolution electrophoresis, and powerful be as trustworthy, as is generally accepted, as a
pattern recognition and clustering software are needed confirmatory tool for species identification. In ad-
to automate species identification based on tDNA- dition, PCR-based DNA fingerprinting enabled the
spacer-PCR. identification of four rarely encountered biochemical
In addition to biochemical characterization, Listeria reaction patterns in L . rnonocytogenes and L. seeligeri
isolates may also be differentiated on the basis of their (indicated as a-d in Table 1).
antigenic composition. Thirteen somatic antigens and Because of discrepancies between 16s ARDRA results
five flagellar antigens have been described in the and computer-assisted restriction analysis of the pub-
Listeria species and are currently used for serotyping lished sequences (7,8), the sequences of the 1 s rRNA
(35). However, undescribed combinations of antigens genes of seven representative isolates were determined
are regularly encountered during serotyping of field again. Computer-assisted restriction analysis of the
isolates, several of which are listed in the present work. sequences as determined in this study was almost in
Some but not all serovars are species-specific. On one complete agreement with ARDRA results (Table 2),
136 International Journal of Systematic Bacteriology 48
PCR fingerprinting for identification of Listeria species
thus confirming the validity of the new updated rRNA the reliable identification of biochemically and sero-
gene sequences. logically atypical Listeria isolates. The data reported
Besides possible sequencing errors, other reasons have here confirm a high genetic relatedness between most
been discussed previously (6) and may explain the Listeria species and suggest a closer relationship than
discrepancies between the three independant 16s expected between the two non-haemolytic species L.
rRNA gene sequence determinations carried out thus innocua and L. welshimeri, which also share common
far (7, 8, this study). Furthermore, application of serotypes. The combination of the 16s rDNA sequence
reverse transcription prior to sequencing may be a data and the data gathered with the different PCR
major source of sequence errors. fingerprinting methods suggests that L. innocua is as
closely related to L. welshimeri as it is to L. mono-
The results obtained here with different PCR-based cytogenes and confirms that there are subtle but
DNA fingerprinting techniques reflect the previously consistent genetic differences between the two L.
established high intragenic relatedness of the Listeria ivanovii subspecies and that L. grayi is homogeneous
species. For example, for a total of 17 restriction and is validly included in the genus Listeria.
enzymes out of 22 tested, not a single restriction site
difference could be established in the 16s rDNA of the
six species. Correspondingly, the new sequences pre- ACKNOWLEDGEMENTS
sented here indicate that the dissimilarity estimates We are very grateful to C . L. Gyles for checking the
between the 16s rRNA genes of the different Listeria manuscript.
species are even lower than previously reported (7),
although this does not influence the phylogenetic
relatedness as has been established previously. REFERENCES
Subtle, but reproducible, differences between L. 1. Bannerman, E., Yersin, M. N. & Bille, 1. (1992). Evaluation of
ivanovii subsp. ivanovii and L. ivanovii subsp. london- the Organon-Technika MICRO-ID LISTERIA system.
iensis could be established by several techniques. Appl Environ Microbiol58, 201 1-2015.
Sequence comparison of the first 1200 bp of the 16s 2. Bille, J., Catimel, B., Bannerman, E., Jacquet, C., Yersin, M. N.,
rDNA revealed only two base pair substitutions (at Caniaux, I., Monget, D. & Rocourt, J. (1992). API Listeriu, a
positions 46 and 1096) between both subspecies, and new and promising one-day system to identify Listeriu
isolates. Appl Environ Microbiol58, 1857-1 860.
single bp length differences were observed between
tDNA spacers of isolates of both subspecies. These 3. Boerlin, P., Rocourt, J., Grimont, F., Grimont, P. A. D., Jacquet,
C. & Piffaretti, I.-C. (1992). Listeriu ivunovii subsp. london-
data fit the classification of the two corresponding
iensis subsp. nov. Int J Syst Bucteriol42, 69-73.
genomic groups at the subspecies level (3) and not as
different species. Only ARDRA and tRNA-spacer- 4. Boerlin, P., Rocourt, 1. & Piffaretti, I.-C. (1991). Taxonomy of
PCR could establish some differences between L. the genus Listeriu by using multilocus enzyme electro-
phoresis. Int J Syst Bucteriol41, 59-64.
innocua and L. welshimeri. Interestingly, L. innocua
and L. welshimeri also share common serotypes. 16s 5. Cartwright, C. P., Stock, F., Beekmann, 5. E., Williams, E. C. &
Gill, V. J. (1995). PCR amplification of rRNA intergenic
rDNA sequence similarity distances as calculated here
spacer regions as a method for epidemiological typing of
for the species pairs L. monocytogenes - L. innocua Clostridium dificile. J Clin Microbiol33, 184-1 87.
and L. welshimeri L. innocua are comparable (Table
3). Finally, the fingerprints obtained for L. grayi 6. Clayton, R. A., Sutton, G., Hinkle, P. S., Jr, Bult, C. & Fields, C.
(1995). Intraspecific variation in small-subunit rRNA se-
differed most from those of all the other Listeria quences in GenBank. Why single sequences may not
species, thus confirming its distance from the rest of adequately represent prokaryotic taxa. Int J Syst Bucteriol
the genus (4, 7, 12, 31). L. grayi was found to be 45,595-599.
homogeneous with all techniques (except with rDNA- 7. Collins, M. D., Wallbanks, S., Lane, D. J., Shah, J., Nietupski,
spacer-PCR), which is in agreement with the recent R., Smida, J., Dorsch, M. & Stackebrandt, E. (1991). Phylo-
grouping of L. grayi and ‘ L .murrayi’ into one unique genetic analysis of the genus Listeriu based on reverse
species (33). transcriptase sequencing of 16s rRNA. Int J Syst Bucteriol
Conclusions 8. Czajka, J., Bsat, N., Piani, M., Russ, W., Sultana, K., Wiedmann,
M., Whitaker, R. & Batt, C. A. (1993). Differentiation of
In conclusion, the previously published 16s rRNA Listeriu monocytogenes and Listeriu innocuu by 16s rRNA
gene sequences of all six Listeria species have been genes and intraspecies discrimination of Listeriu mono-
updated and now fit the restriction profiles obtained by cytogenes strains by random amplified polymorphic DNA
ARDRA. In our hands, ARDRA performed best with polymorphisms. Appl Environ Microbiol59, 304308.
respect to both discriminatory power and practical 9. Deng, S., Hiruki, C., Robertson, 1. A. & Stemke, G. W. (1992).
applicability. Satisfactory results were obtained with Detection by PCR and differentiation by restriction frag-
rDNA-SSCP analysis and tDNA-spacer-PCR, al- ment length polymorphism of Acholeplusmu, Spiroplusmu,
though the former requires thorough standardization Mycoplusmu, and Ureuplusmu, based upon 16s rRNA
of electrophoresis conditions and the use of additional genes. PCR Methods Appl 1,202-204.
primer sets, and the latter requires high-resolution 10. Ehrenstein, B., Bernards, A. T., Dijkshoorn, L., Gerner-Smidt,
electrophoresis. All of these techniques also allowed P., Towner, K. J., Bouvet, P. 1. M., Daschner, F. D. & Grund-
International Journal of Systematic Bacteriology 48 137
M. Vaneechoutte and others
mann, H. (1996). Acinetobacter species identification by 25. Kostman, J. R., Edlind, T. D., Lipuma, J. 1. & Stull,T. L. (1992).
using tRNA spacer fingerprinting. J Clin Microbiol 34, Molecular epidemiology of Pseudomonas cepacia deter-
24 14-2420. mined by polymerase chain reaction ribotyping. J Clin
11. Fernandez-Garayzabal, J. F., Suarez, G., Blanco, M. M., Microbiol30, 20842087.
Gibello, A. & Dominguez, L. (1996). Taxonomic note: a 26. McClelland, M., Petersen, C. & Welsh, 1. (1992). Length
proposal for reviewing the interpretation of the CAMP polymorphisms in tRNA intergenic spacers detected by
reaction between Listeria monocytogenes and Rhodococcus using the polymerase chain reaction can distinguish strepto-
equi. Int J Syst Bacteriol46, 832-834. coccal strains and species. J Clin Microbiol30, 1499-1504.
12. Feresu, 5. B. & Jones, D. (1988). Taxonomic studies on 27. Michel, E. & Cossart, P. (1992). Physical map of the Listeria
Brochothrix, Erysipelothrix, Listeria, and atypical lacto- monocytogenes chromosome. J Bacterioll74, 7098-7 103.
bacilli. J Gen Microbiol 134, 1165-1 183. 28. Nesme, X., Vaneechoutte, M., Orso, S., Hoste, B. & Swings, J.
13. Gilrtler, V., Wilson, V. A. & Mayall, B. C. (1991). Classification (1995). Diversity and genetic relatedness within genera
of medically important clostridia using restriction endo- Xanthomonas and Stenotrophomonas using restriction endo-
nuclease site differences of PCR-amplified 16s rDNA. J nuclease site differences of PCR-amplified 16s rDNA. Syst
Gen Microbioll37, 2673-2679. Appl Microbiol18, 127-135.
14. Gurtler, V. (1993). Typing of Clostridium dzjicile strains by 29. Ralph, D., McClelland, M., Welsh, J., Baranton, G. & Perolat,
PCR-amplification of variable length 16s-23s rDNA spacer P. (1993). Leptospira species categorized by arbitrarily
regions. J Gen Microbioll39, 3089-3097. primed polymerase chain reaction (PCR) and by mapped
15. GUrtler, V. & Stanisch, V. A. (1996). New approaches to restriction polymorphism in PCR-amplified rRNA genes. J
typing and identification of bacteria using the 16s-23s Bacterioll75, 973-98 1.
rDNA spacer region. Microbiology 142, 3-16. 30. Rocourt, J., Grimont, F., Grimont, P. A. D. & Seeliger, H. P. R.
16. Heyndrickx, M., Vandemeulebroecke, K., Scheldeman, P., (1982). DNA relatedness among serovars of Listeria mono-
Hoste, B., Kersters, K., De Vos, P., Logan, N. A., Aziz, A. M., cytogenes sensu lato. Curr Microbiol7, 383-388.
Ali, N. & Berkeley, R. C. W. (1995). Paenibacillus (formerly 31. Rocourt, J., Schrettenbrunner, A. & Seeliger, H. P. (1983).
Bacillus) gordonae (Pichinoty et al. 1986) Ash et al. 1994 is Diffirenciation biochimique des groupes ginomiques de
a later subjective synonym for Paenibacillus (formerly Listeria monocytogenes (sensus lato). Ann Microbiol Inst
Bacillus) validus (Nakamura 1984) Ash et al. 1994: emended Pasteur 134A, 65-7 1.
description of P. validus. Int J Syst Bacteriol45, 661-669. 32. Rocourt, J., Wehmeyer, U., Cossart, P. & Stackebrandt, E.
17. Hookey, 1. V., Birtles, R. 1. & Saunders, N. A. (1995). Inter- (1987). Proposal to retain Listeria murrayi and Listeria grayi
genic 16s rRNA gene (rDNA)-23S rDNA sequence length in the genus Listeria. Int J Syst Bacteriol37, 298-300.
polymorphisms in members of the family Legionellaceae. J 33. Rocourt, J., Boerlin, P., Grimont, F., Jacquet, C. & Piffaretti, 1.-
Clin Microbiol33, 2377-238 1. C. (1992). Assignment of Listeria grayi and Listeria murrayi
18. Jannes, G., Vaneechoutte, M., Lannoo, M., Gillis, M., Van to a single species, Listeria grayi, with a revised description
Canneyt, M., Vandamme, P., Verschraegen, G., Van of Listeria grayi. Int J Syst Bacteriol42, 171-174.
Heuverswyn, H. & Rossau, R. (1993). Polyphasic taxonomy 34. Sallen, B., Rajoharison, A., Desvarenne, S., Quinn, F. &
leading to the proposal of Moraxella canis sp. nov. for Mabilat, C. (1996). Comparative analysis of 16s and 23
Moraxella catarrhalis-like strains. Int J Syst Bacteriol 43, rRNA sequences of Listeria species. Int J Syst Bacteriol46,
19. Jayarao, B. M., Dore, 1. J. E., Jr, Baumbach, G. A., Matthews, 35. Seeliger, H. P. R. & Htihne, K. (1979). Serotyping of Listeria
K. R. & Oliver, 5. P. (1992). Restriction fragment length monocytogenes and related species. Methods Microbiol 13,
polymorphism analysis of 16s ribosomal DNA of Strep- 3 1-49.
tococcus and Enterococcus species of bovine origin. J Clin
Microbiol30, 2235-2240. 36. van Belkum A., Kluytmans, J., van Leeuwen, W. &13 other
authors (1995). Multi-center evaluation of arbitrarily
20. Jensen, M. A., Webster, 1. A. & Straus, N. (1993). Rapid primed PCR for typing of Staphylococcus aureus strains. J
identification of bacteria on the basis of polymerase chain Clin Microbiol33, 1537-1 547.
reaction-amplified ribosomal DNA spacer polymorphisms.
Appl Environ Microbiol59, 945-952. 37. Vaneechoutte, M., Cartwright, C. P., Williams, E. C., Jlger, B.,
Tichy, HA., De Baere, T., De Rouck, A. & Verschraegen, G.
21. Jensen, M. A. & Straus, N. (1993). Effect of PCR conditions (1996). Evaluation of 16s rRNA gene restriction analysis
on the formation of heteroduplex and single-stranded DNA for the identification of cultured organisms of clinically
products in the amplification of bacterial ribosomal DNA important Clostridium species. Anaerobe 2, 249-256.
spacer regions. PCR Methods Appl3, 186-194.
38. Vaneechoutte, M., De Beenhouwer, H., Claeys, C.,
22. Klmpfer, P., Bt)ttcher, S , Dott, W. & RUden, H. (1991).
. Verschraegen, G., De Rouck, A., Paepe, N, Elaichouni, A. &
Physiological characterization and identification of Listeria Portaels, F. (1993). Identification of Mycobacterium species
species. Int J Med Microbiol275, 423435. with amplified rDNA restriction analysis. J Clin Microbiol
23. Kathariou, S. & Pine, L. (1991). The type strain(s) of Listeria 31,2061-2065.
monocytogenes : a source of continuing difficulties.Int J Syst 39. Vaneechoutte, M., Dijkshoorn, L., Tjernberg, I., Elaichouni,
Bacteriol41, 328-330. A., De Vos, .
P, Claeys, G. & Verschraegen, G. (1995).
24. Kerr, K. G, Rotowa, N. A., Hankey, P. M. & Lacey, R. W.
. Identification of Acinetobacter genomic species by amplified
(1991). Evaluation of the ROSCO system for the iden- ribosomal DNA restriction analysis. J Clin Microbiol 33,
tification of Listeria species. J Med Microbiol35, 193-196. 11-15.
138 International Journal of Systematic Bacteriology 48
PCR fingerprinting for identification of Listeria species
40. Vaneechoutte, M., Riegel, P., de Briel, D., Monteil, H., polymorphisms in tRNA intergenic spacers for categorizing
Verschraegen, G., De Rouck, A. & Claeys, G. (1995). staphylococci. Mol Microbiol6, 1673-1680.
Evaluation of the applicability of amplified rDNA-restric- 44. Widjojoadmotjo, M. N., Fluit, A. C. & Verhoef, 1. (1994).
tion analysis to identification of species of the genus Rapid identification of bacteria by PCR-single-strand
Corynebacterium. Res Microbioll46, 633-641. conformation polymorphism. J Clin Microbiol 32,
41. Vaneechoutte, M., Rossau, R, DeVos, P., Gillis, M., Janssens,
D., Paepe, N., De Rouck, A., Fiers, T., Claeys, G. & Kersters, K. 45. Widjojoadmotjo, M. N., Fluit, A. C. & Verhoef, J. (1995).
(1992). Rapid identification of bacteria of the Com- Molecular identification of bacteria by fluorescence-based
amonadaceae with amplified ribosomal DNA-restriction PCR-single-strand conformation polymorphism analysis of
analysis (ARDRA). FEMS Microbiol Lett 93, 227-234. the 16s rRNA gene. J Clin Microbiol33, 2601-2606.
42. Welsh, J. & McClelland, M. (1991). Genomic fingerprints 46. Wiedmann-Al-Ahmad, M., Tichy, H A . & Schan, G. (1994).
produced by PCR with consensus tRNA gene primers. Characterization of Acinetobactev type strains and isolates
Nucleic Acids Res 19, 861-866. obtained from wastewater treatment plants by PCR finger-
43. Welsh, J. & McClelland, M. (1992). PCR-amplified length printing. Appl Environ Microbiol60, 4066-4071.
International Journal of Systematic Bacteriology 48 139